Moraxella catarrhalis outer membrane protein CD elicits antibodies that inhibit CD binding to human mucin and enhance pulmonary clearance of M. catarrhalis in a mouse model

Abstract

The outer membrane protein CD of Moraxella catarrhalis is considered to be a potential vaccine antigen against Moraxella infection. We purified the native CD from isolate O35E, administered it to mice, and detected considerable titers of anti-CD antibodies. Anti-CD sera were cross-reactive towards six different M. catarrhalis isolates and promoted bacterial clearance of O35E in a pulmonary challenge model. To circumvent the difficulty of generating large quantities of CD from M. catarrhalis for vaccine use, the CD gene from O35E was cloned into Escherichia coli, and the recombinant CD, expressed without a signal sequence or fusion tags, represented approximately 70% of the total E. coli proteins. The recombinant CD formed inclusion bodies that were solubilized with 6 M urea and then purified by ion-exchange chromatography, a procedure that produced soluble CD of high purity and yield. Mice immunized with the purified recombinant CD had significant titers of anti-CD antibodies that were cross-reactive towards 24 different M. catarrhalis isolates. Upon challenge, these mice showed enhanced bacterial clearance of both O35E and a heterologous M. catarrhalis isolate, TTA24. In an in vitro assay, antisera to either the native or the recombinant CD inhibited the binding activity of CD to human tracheobronchial mucin in a serum concentration-dependent manner, and the extent of inhibition appeared to correlate with the corresponding anti-CD antibody titer and whole-cell enzyme-linked immunosorbent assay titer. Our results demonstrate that the recombinant CD is a promising vaccine candidate for preventing Moraxella infection.

FIG. 1.

Enhanced pulmonary clearance of M. catarrhalis isolate O35E in mice immunized with nCD. Mice immunized twice with 5 μg of nCD with MPL plus alum as an adjuvant were challenged intratracheally with 3.5 × 10⁵ CFU of O35E. Six hours after challenge, CFU recovered from the lungs of individual mice were determined. Each bar represents the standard deviation of the mean. *, P = 0.0007 compared to the sham group CRM197 + MPL/alum.

FIG. 2.

SDS-PAGE analysis of fractions obtained at different stages of rCD purification. A 12% Coomassie blue-stained SDS gel was loaded with 5 μg of protein in each lane. Lane 1, Bio-Rad prestained low-molecular-mass markers in kDa; lane 2, cell lysate of an induced culture of E. coli containing pLP150; lane 3, rCD inclusion bodies solubilized with 6 M urea; lane 4, rCD eluted off the TMAE column; lane 5, rCD eluted off the SP column and then dialyzed into PBS containing 0.1% Triton X-100.

FIG. 3.

Enhanced pulmonary clearance of M. catarrhalis isolates O35E and TTA24 in mice immunized with rCD. Mice immunized twice with 20 μg of rCD mixed with different adjuvants were challenged intratracheally with either O35E or TTA24 as described in the Fig. 1 legend. (A) Pulmonary clearance of O35E. *, P = 0.012 compared to the sham group CRM197 + alum; **, P = 0.0095 compared to the sham group CRM197 + QS21. (B) Pulmonary clearance of TTA24. *, P = 0.0008, and **, P = 0.0023, compared to the sham group CRM197 + alum. Each bar represents the standard deviation of the mean.

FIG. 4.

(A) Binding to human tracheobronchial mucin as a function of protein concentration of rCD (⧫), rCopB (○), or bovine serum albumin (▵). (B) Inhibition of rCD-mucin binding by anti-rCD serum. The extent of rCD-mucin binding was measured with rCD at 2.5 μg/ml previously incubated with either preimmune serum (⋄) or anti-rCD serum (⧫). The lowest serum dilution was 1:100 and the highest 1:12,800. For each data point, the bar represents the standard deviation of the mean.